The advent of compounds that are generated using recombinant DNA technology has facilitated development of a vast array of therapeutic agents having the potential to treat a great variety of disease states in animals, in particular in humans. These agents are primarily protein in nature.
The epidermis, the squamous stratified epithelium of the skin, consists of multiple sublayers and is one of the most important barriers of the body against the outside world. The stratum corneum is the outermost layer of the epidermis and develops as a result of the final anucleated step in keratinocyte differentiation from the cells in nucleated epidermal layers. Although the stratum corneum is recognized as the most important physical barrier, the nucleated epidermal layers are also significant in barrier function (Proksch. Brandner et al. 2008). Together, the skin barrier protects against extensive water loss in one direction (inside-outside barrier) and against the invasion of harmful substances from the environment (outside-inside barrier) (Proksch, Brandner et al. 2008). The maintenance of the barrier is also important for balanced proliferation in the basal layer and preservation of the calcium ion gradient and thus proper epidermal differentiation (Lee, Jeong et al. 2006).
Recent work suggests that skin commensal microorganisms are essential to maintaining healthy skin and maintaining the skin barrier. Commensal microorganisms are ones that are beneficial to their subject. e.g., human, hosts. Studies also suggest that certain skin diseases (such as acne vulgaris and atopic dermatitis) can be associated with disruptions to the normal microflora (Lin, Wang et al. 2007). Therefore, the idea that the skin microflora can be modulated using specific skin commensals to promote health or inhibit disease has received some attention (Muizzuddin, Maher et al. 2012). Many skin commensal bacteria are not considered pathogenic, therefore, these bacteria can potentially be used topically if they have therapeutic value (Nakatsuji and Gallo 2014). So far the limited amount of research in this area suggests that conventional probiotic bacteria can be of significant value when used on the skin. For example, topical application of a Bifidobacterium longum lysate has been shown to induce clinical improvement of “reactive skin” (Gueniche, Bastien et al. 2010). This is skin that is more sensitive to physical changes such as atmospheric temperature, and to chemical changes such as seen with topically applied products (Gueniche, Bastien et al. 2010). Application of the B. longum lysate to the skin of volunteers was shown to decrease sensitivity and decrease transepidermal water loss (TEWL) after tape stripping. Additionally, application of the lysate to ex vivo skin was shown to decrease signs of inflammation such as vasodilation, edema and TNF-α release (Gueniche, Bastien et al. 2010, Nakatsuji and Gallo 2014, Volz, Skabytska et al. 2014). Topical application of Lactobacillus plantarum has also been demonstrated to improve tissue repair in a burned mouse model and prevent infection in chronic leg ulcers and burns in humans (Peral. Martinez et al. 2009, Peral, Rachid et al. 2010, Brachkova, Marques et al. 2011).
A number of current limitations exist in the treatment of skin, however. Many treatments, such as topical corticosteroids or expensive biologics, do not treat the underlying issues of deficient intrinsic protein in the epidermis or imbalances in the microbial diversity in the skin. While recombinant proteins represent a promising group of therapeutic agents in the treatment of skin disease, several problems accompany their use in the context. Traditional methods employ the use of purification of recombinant proteins that are extracted from bacterial systems, purified, concentrated, and incorporated into a delivery system. The purification of recombinant proteins is often a very costly method of obtaining protein. Moreover, a number of issues accompany this, including proteolytic degradation, inefficient delivery, and the need for repeated application overtime to achieve therapeutic effect.
Our method, in contrast, addresses these issues by allowing bacteria to colonize the skin and continually secrete therapeutic polypeptides of benefit to the skin. This approach reduces need for multiple topical applications by creating a sustainable delivery system for therapeutic proteins.
Most of the microorganisms until now used for the production of recombinant proteins cannot be in safely administered to humans and animals, not being usual components of the physiological flora or being devoid of any pathogenic risk. This is particularly true for Escherichia coli, which is considered pathogenic in many cases. However, here, we describe methods of production of recombinant proteins using microorganisms that can be safety administered to humans and animals, particularly Staphylococcus epidermidis. 
Other species, already used for the production of recombinant proteins, can also be used provided they meet the requisites of non-pathogenicity and ability to colonize human or animal mucosae. For example, Bacillus subtilis has been widely used as a cloning vector for producing a large number of eukaryotic proteins in view of its recognized advantageous properties (Simonen and Palva 1993). The present invention allows therefore, by suitably selecting and adapting the microorganism, the polypeptide to be expressed and the expression vectors, previously used for the production in laboratory or industrial environment, to address specific skin diseases (see FIG. 1). The present invention concerns therefore the therapeutic use of said engineered microorganisms and compositions containing the same, thereby satisfying a long felt need in the area of therapeutic delivery of drugs to treat skin conditions in humans.
Atopic dermatitis (AD), which is also known as eczema, is a chronic inflammatory skin disease that usually involves a defect in the stratum corneum of the skin, which is the protective layer comprising the outermost part of the skin. This defect is caused by a confluence of genetic, environmental, and immunological factors and is associated with an overgrowth of Staphylococcus aureus. The genetic basis of AD appears to involve a defect in the filaggrin gene (FLG), seen in approximately 50% of AD patients (McAleer 2013). The prevalence of atopic dermatitis is increasing, and affects 0.1-1% of the population worldwide, with higher rates in developed countries. AD is more common in children, affecting as many as 20% of children (McAleer 2013). Of these children, 25% never recover and continue to suffer from atopic dermatitis into adulthood. In many cases, AD can lead to more severe allergic conditions, including asthma, through a process known as the “atopic march.”
The pathogenesis of AD is not completely understood, but defects in filaggrin expression are attributed to most cases of atopic dermatitis, and the pathogenesis is related to an immune response. It has been shown that AD can be caused by a combination of dry skin, skin that is prone to itching more than the average person, infections caused by bacteria, viruses, fungi, etc., and emotional and environmental factors. Filaggrin is encoded by the FLG gene on the 1q21 epidermal differentiation complex. Filaggrin is a protein produced by differentiating keratinocytes, and functions to aggregate keratin filaments into a cytoskeleton that, in combination with other components, comprises the cornified cell envelope (Brown & McLean 2012). Filaggrin loss of function mutations (R501X and 2282del4) cause ichthyosis vulgaris, the most common inherited disorder of keratinization. The same mutations also are associated with other skin and allergic conditions including AD, irritant contact dermatitis, asthma, and food allergy. Several studies have shown that increasing filaggrin on the skin helps to mitigate the AD phenotype (e.g. Stout et al. 2014, Otsuka et al. 2014).
Currently, the major therapies for AD include topical corticosteroids and antibiotics, both of which have limited efficacy in more severe cases of AD. Major drawbacks of antibiotics include issues of antibiotic resistance and dysregulation of the microbiome ecology. The microbiome is the aggregate of microorganisms present in a particular environment. The microbiome mentioned herein, is the totality of the microorganisms present on the largest human organ, the skin. Topical corticosteroids provide respite from symptoms for mild AD patients, but do not address the needs of those with chronic or severe disease. While some targeted therapies for AD treatment are under development, the competitive landscape in this market is characterized by a largely unmet need for an effective, targeted therapy for AD. One biologic therapy, Dupilumab, is an anti-IL4/13-receptor monoclonal antibody that is under Phase II clinical trial for AD, being developed by Regeneron Pharmaceuticals and Sanofi. This drug has shown promising efficacy, and is expected to reach markets in the next five years. Other similar biologic therapeutics for immunomodulation of AD are under development. However, these drugs are expected to cost tens of thousands of dollars for consumers due to high production and R&D costs, and insurance companies may not necessarily cover these costs. While these drugs attenuate the immune response, they do not have direct effects on re-establishing the skin barrier and normalizing the microbial ecology to prevent S. aureus infections. Thus, new compositions and methods for the effective treatment and prevention of abnormal skin conditions such as AD, while minimizing unwanted side effects are required.